I. Field of the Invention
The present invention relates to methods and apparatus for assessing the hepatotoxicity of a stimulus. More particularly, the present invention relates to image analysis methods and apparatus that characterize a stimulus based upon phenotypic characteristics of hepatocytes and some other cell types exposed to the stimulus.
II. Background
Hepatotoxicity is a major safety concern for drug development. Approximately 90 percent of lead candidates fail to become drugs, and hepatotoxicity accounts for about 22 percent of these failures. Traditionally, a variety of strategies have been used to predict hepatotoxicity during preclinical development. These include incubating compounds with cultured hepatocytes to measure cytotoxicity or induction of the various isoforms comprising the drug metabolizing CYP enzymes. Biochemical enzyme assays, using purified CYP enzymes or crude liver microsome extracts, are used to determine the substrate activities of drug candidates and to profile their metabolic products using chromatographic methods.
Animal studies have also been widely used to predict human hepatotoxicity. In these studies, rats or mice are dosed with various concentrations of the test compound, and the animals are monitored for important serum markers such as serum albumin, prothrombin, bilirubin, AST, ALT, and alkaline phosphate at different time points. The animals are then sacrificed, and a full histopathological analysis of the liver, kidney, and other important organs and/or tissues is carried out.
More recently, gene expression studies have been used to predict hepatotoxicity. The RNA is isolated from cultured hepatocytes or liver sections from animals and analyzed using microarray technology. The advantages of this approach include faster turnaround times and less labor compared to animal pathology studies, and the method (as applied to cultured hepatocytes) requires a smaller amount of the experimental compound. Many industrial and academic groups are attempting to identify key genes that are expressed during a hepatotoxic response. The goal is to create a database that contains the gene expression patterns of known hepatotoxins and associated liver pathologies. The database is then used to predict the mechanism of hepatotoxicity by comparing the gene expression patterns of a new compound to those of reference compounds. This approach is still under development, and the number of marker genes reported to be relevant for the rat model varies from 400 to over 3000 genes. See “Serious liver injury: leading reasons for drug removals, restrictions” www.fda.gov/fdac/features/2001/301_liver.html; and ToxExpress™ Application Note, GeneExpress ToxExpress Predictive System™, Gene Logic, Gaithersburg, Md. (2002).
Unfortunately, none of the traditional approaches adequately predicts the hepatotoxic potential of drugs that reach the marketplace. At least three drugs within the last five years, Duract (bromfenac), Trovan (trovafloxacin) and Rezulin (troglitazone), have significant use limitations or were pulled from the market due to human hepatotoxicity. Thus, there is a great need for new methods to predict hepatotoxicity, and to use these methods early in the lead optimization process to save time and cost.